Protein degradation plays an important role in the regulation of growth and atrophy of mammalian tissues. However, the exact enzymes which catalyze protein degradation in vivo are not clearly defined nor is it clear how their activities are regulated. Rat liver cytoplasm contains two groups of recently discovered endoproteases which are regulated in vitro by physiologically important molecules. The first group consists of two high molecular weight, ATP-stimulated proteases (Mr equals 750,000 and Mr equals 550,000). The second group consists of two smaller proteases (Mr equals 150,000) which are totally dependent on micromolar concentrations of calcium for activity. The specific aims of this research are 1) to purify each of these proteases 2) to characterize each of the purified enzymes with respect to physical and enzymatic properties, especially the possible regulatory mechanisms involved with ATP and calcium 3) to identify and examine differences and possible similarities in the structure and enzymatic function among these various enzymes. To achieve these goals, we will employ biochemical approaches including modern protein purification techniques and physical kinetic methods in enzymology. Information gained in this study will relate directly to our long-term goals of identifying proteases in mammalian cells and defining their roles in and regulation of the degradation of endogenous cell proteins.